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Isolation of haustoria from wheat leaves infected by the leaf rust fungus
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PItvsiological and Molecular Plant Pathology 11993/ 42, 337 343
Isolation of haustoria from wheat leaves infected by the leaf rust fungus L. C. CANTRILL and B.J. DEVERALL D@aHment of Cr@ Sciem:e.~, t 'niversity of Sydney, . V.S. [ I ". 2006, Australia .lccepted Jar publication .l larch 19931
A proeedure was developed to isolate haustoria from wheat leaves infected by Puccinia recondita. Homogenization of heavily inR'cted leaves in an isolation medium ,.,.'as followed bv a period of sedimentation. 'fhe resulting suspension,,,,'assubjected to mild centrifugation and the pellet was separated further by centrifugation on a density gradient. One laver ',','asselected and washed before examination by optical microscopy. Two types of fungal structure were distinguishcd by their shape, size and cytoplasmicactivity and with a nuclear stain and Icctin probcs. One type was identified as hyphae and the other as haustoria. The rcasons for identification arc discussed in relation to literature on isolated haustorial complexes fi'om powdery mildew fungi and on clcctron microscopic examination of sections of rust haustoria in wheat leaves.
INTRODUCTION Haustoria are the only structures of rust and powdery mildew fungi that come into direct contact with the host protoplasm [1] and for this reason they are likely sites for the initiation of mechanisms of resistance and specificity. Access to haustoria for physiological and molecular studies is needed. I n rust fungi, the haustoria are e m b e d d e d deeply in the host tissue and m u c h has been written c o n c e r n i n g the difficulties thus posed in working with them [7]. In vitro formation of haustoria [10, I1 ] a n d isolation and purification of haustoria or haustorial complexes from the leaf [5] offer approaches in yielding haustoria that are free of host tissue. T h e a p p r o a c h of isolating rust haustoria and hyphae from leaves has been used here along with methods for confirming the n a t u r e of the fungal parts so obtained. Detailed ultrastructural and cytochemical analyses of the hyphae and haustoria of the wheat stem rust fungus have been achieved using lectin stains applied to c o n v e n t i o n a l electron microscopy preparations [3, 4 ]. A model of the c o m p o n e n t layers in rust fungal walls has been derived from the use of these stains. C h o n g et al. [4] represented haustorial walls with two layers, both of which contained chitin. H y p h a l walls (containing four layers) and haustorial m o t h e r cell walls (containing up to six layers) appeared to possess no chitin in outer layers [3], but carried surface residues of an a-linked glucose or m a n n o s e which were not detected in haustorial walls. T h u s a possible cytochemical method for differentiating haustorial surfaces from hyphal surfaces was indicated. Recent i m p r o v e m e n t s to p r e p a r a t i o n techniques for electron microscopy have shown 0885-5765/93/050337 + 07 $08.00/0
~) 1993 Academic Press Limited
338
L.C. Cantrill and B. J. Deverall
the cytochemistry of rust fungal walls to be even more complex, involving previously undetected lectin binding sites [13] and variable lectin binding in the extramural environment [9]. While such improvements refine the knowledge of non-specific lipids, polysaccharides and glycoproteins in and around the walls, the results caution against reliance upon lectin staining for distinguishing haustorial and hyphal surfaces. MATERIALS AND METHODS
Infected plants Seeds of Triticum aestivum L. cv. Little Club (10 per 12"5-cm pot in 650 g of a peat/river sand mixture with complete fertilizer) were germinated at approximately 22 °C under 16-h photoperiods (250 p.Em -2 s-1 at soil level). At 8 days, primary leaves of 30 plants were inoculated with 100 mg of washed uredospores of Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. & Henn. (race 104-2,3,6,7) by applying a spore/talc mixture (6: 10) using a paint brush. Plants were incubated in a dark humid chamber for 18 h at 23 °C (overnight) and then were returned to the conditions of seed germination for 7-14 days.
Phase contrast microscopy Heavily infected primary leaves (1"5 g) were homogenized for l min in 10 ml of isolation medium (0-1 M sucrose/0"01 M phosphate buffer pH 6"7, adapted from Gil & Gay [5]) using a Sorvall Omnimix set at speed 6. The homogenate was fhered through muslin and allowed to stand for 30 min in order to remove spores and other large material. The filtrate was then decanted from the precipitate and saved. Homogenization of retained debris was repeated three times (speed set successively to 6, 7 and 8) with further filtering and collection of filtrate after each speed increment. Each sample of filtrate was centrifuged at 800 g for 10 min. The supernatant was discarded and the pellet resuspended in 2 ml isolation medium. Drops of the suspension were placed on slides and examined with phase contrast on a Leitz Dialux microscope. Fluorescence of nuclei The cellular contents of the fungal material in resuspended pellets were examined using bisbenzimide H33258 fluorochrome (Hoechst). The dye was prepared by adding 0"6 ml of a stock solution (20 mg of Hoechst 33258 in 25 ml of distilled water) to 50 ml of isolation medium (adapted from Hua'an [12]). Dye solution (1 ml) was added to 1 ml offungal suspension. Mounts of whole mycelia were also prepared for comparison with the homogenized material. Squares (5 x 5 mm) of infected wheat leaf were incubated for 3 h in a macerating solution (0"5 °/o pectinase, 1"0 °/o hemicellulase, 2"0 °/o cellulase in a 10% sorbitol solution) and teased apart on a slide in a drop of dye solution. Both preparations were examined under UV illumination using a Leitz Orthoplan microscope fitted with a 200 W high-pressure mercury lamp and Leitz type A filter blocks. Fluorescent lectins Suspensions of fungal material were prepared as for phase contract microscopy. Each 2 ml suspension was layered onto an 8 ml 10-20 % continuous Ficoll 400 (Pharmacia) gradient made up in isolation medium and centrifuged for 5 min at 100 g. The middle
Isolation of haustoria
339
greenish-white layer of each gradient was collected and washed by diluting with isolation medium and centrifuged at 800 g for 10 min. The supernatant was discarded and the washing step repeated on the pelleted material. The final pellets were resuspended in 1 ml of the appropriate agglutinin buffer and 1 ml of stock agglutinin was added. The surfaces of the extracted fungal material were examined with the aid of either concanavalin agglutinin (ConA) or wheat germ agglutinin (WGA), each labelled with ftuorescein isothiocyanate (FITC). FITC--ConA was dissolved in a solution of 0"01 M Tris-HC1, 0"15 M NaC1, 1 mM CaCI~, and 1 mM MnC12 at p H 7"2 to make a final concentration of 200 lag ml -x in the fungal suspension. F I T C - - W G A was dissolved in 0-1 M phosphate buffer p H 7"2 to make a final concentration of 100 lag m1-1 in the fungal suspension (adapted from Chard & Gay [2]). Drops of the stained fungal suspension were mounted on slides and fluorescence was observed under UV with Leitz type I and 12 filter blocks. RESULTS
Homogenization released a mixture of fragments into the isolation medium (Fig. 1). Partial purification of the crude extract on a Ficoll 400 gradient eliminated most of the contaminating chloroplasts and removed the majority ofuredospores and larger fungal material. Two classes of structure remained and these were identified as hyphal fragments and haustoria for the following reasons. Hyphal fragments (Figs 1 and 4) were 4"5-5 lam wide and irregular in shape along
F[o. 1. Crude homogenatetreated with FITC-WGA ( 1O0 lag ml-x) showingbrightly fluorescent haustoria (H), chloroplasts (cl) and a large fragment of hypha (h) with septum (s).
340
L.C. Cantrill and B. J. Deverall
their axes, being periodically septate and branched. The ends of the fragments were roughly sheared. No cytoplasmic particles were observed under phase contrast and no Hoechst fluorochrome staining bodies were seen under UV illumination. The walls of hyphal fragments fluoresced lightly under UV illumination after staining with F I T C - C o n A or F I T C - W G A . The other class of fragments, identified as haustoria, were elongate (20-45 gm long, 5-10 gm wide) with rounded ends (Fig. 2). The outer walls were entire and there were
FIG. 2. Isolated haustorium under phase contrast showing neck-like projection (n) and cytoplasmic particles (cp). no septae or branches. Each haustorium bore a single rigid neck-like projection. The majority ofhaustoria viewed with phase contrast had transparent walls through which rapidly streaming cytoplasmic particles (0"5 gm diameter) were observed (Fig. 2). In some haustoria the path of the particles was obscured by an amorphous material attached to the walls. Streaming rates were reduced or stopped during further processing on Ficoll gradients and washing by centrifugation. However, unpurified haustoria continued cytoplasmic streaming for several hours after isolation. After staining with the Hoechst reagent the haustoria were seen to contain single 3 gm diameter spheres that fluoresced bright blue under UV illumination [Fig. 3(b)]. Walls ofhaustoria also fluoresced strongly in areas, usually at one end, when treated with the labelled lectins (Figs 1, 4). The rigid neck-like projection (Figs 2, 3, 4) was located at one end of the haustorium, usually the end that fluoresced least brightly with lectin stains. The projections often fluoresced with either of the two lectin probes (Fig. 4). Under phase contrast (Fig. 2) projections were revealed to be composed of two parallel walls (up to 3-5 gm long) which were continuous with the walls of the main haustorial body. The projections were 1 gm wide where they joined the body, narrowing halfway to 0"5 gm. Hand sections of infected leaves contained intercellular haustoria of the same shape and size as those released by homogenization. Similar haustoria were observed
Isolation of haustoria
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10urn FzG. 3. (a) Bright field view ofhaustorium showing neck-like projection (n), as well as cytoplasmic particles and a large central vacuole (v). (b) Same haustorium treated with Hoechst 33258 fluorochrome and illuminated with UV showing faintly fluorescent walls and brightly fluorescent nucleus (n).
Flo. 4. Haustorium (H) and hyphal fragment (h) treated with FITC-ConA (200 lag ml-t). The neck-like projection (n) is highly visible due to staining with the lectin probe.
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L.C. Cantrill and B. J. Deverall
attached to mycelia released by enzymic digestion of pieces of infected leaves. The nuclei in the haustoria were similar in size and location to the spheres that fluoresced with Hoechst reagent in haustoria in homogenized material. DISCUSSION
Haustoria separated from their host by homogenization were identical in shape to haustoria observed in hand sections and to those attached to whole mycelia released by enzymic digestion of host tissue. The dimensions of the host-free haustoria and of their necks also compared favourably with those shown in sections prepared for electron microscopy [8, 14, 15]. Consistency in size, shape, staining patterns with labelled lectins and possession of neck-like projections on the majority of the isolated haustoria indicated that large numbers of uniform rust haustoria could be separated from wheat leaves by homogenization. The usefulness of these separated haustoria in further research is yet to be determined. Observations of streaming particles within the haustorial cellular space for several hours after isolation indicated that haustoria could be separated with cytoplasmic contents intact. The technique o f H u a ' a n et al. [12] revealed that the cytoplasm of hostfree haustoria contained fluorescent blue nuclei under UV and that these were identical to nuclei observed in hyphae and haustoria of whole mycelia. In contrast, streaming cytoplasm and stainable nuclei in hyphae were lost, probably after breakage or exposure of the protoplast to the isolation medium through wall gaps up to 5 lain wide. Earlier leakage of cytoplasm from haustoria may have been slowed by the narrowness of the space within the neck (0"5 lam) and the length of this narrow channel (3 lam). Gil & Gay [5] explained cytoplasmic retention in powdery mildew haustoria as a consequence of an amorphous plug in the septal pore between the neck and haustorial body and attributed durability to the presence of an extrahaustorial membrane. While rust haustoria do not possess a septum, blockage of the neck space by debris may occur during homogenization. At the same time it is unclear whether an extrahaustorial membrane is present or not. Amorphous material observed on the surface of some haustoria may have been a remnant of the extrahaustorial matrix. The lectin-binding properties of the host-free haustoria produced some conflicting results. From the model of stem rust haustoria described by Chong et al. [3, 4], it might be predicted that only WGA binding sites would be detected if the extrahaustorial membrane and matrix were totally absent. Alternatively, ConA binding sites might indicate the presence of these features. Under the conditions used here, both lectins were observed to bind to the haustoria in a similar pattern. Hyphal fragments also varied in the intensity of fluorescence. This may reflect differences in wall chemistry between leaf and stem rust fungi or differences between fresh and fixed material and the relative preservation and availability of active binding sites. Doubts have also been expressed concerning the reliability of F I T C - W G A and its specificity for chitin [10]. Changes in wall chemistry over time have also been noted, with a loss of ConA binding in hyphae found in sporogenous zones [9] and a changing affinity for ConA and WGA in haustoria as they mature [4]. The variability in lectin binding may have been a consequence of the advanced infections used here and the resultant mix of ages and developmental stages released by homogenization.
Isolation of haustoria
343
After this paper was submitted, the isolation of haustoria of the stem rust fungus of wheat (Puccinia graminis f. sp. tritici) was reported [6, 16] and also of haustoria of three other rust fungi, Puccinia sorghi and two species of Uromyces [6]. The methods involved homogenization of heavily-infected leaves as here, but they were followed by centrifugation on sucrose density gradients [16] or affinity chromatography of ConAbound extracts [6]. Viability of haustoria was recorded to be high according to exclusion of methylene blue stain [16] and to electron microscopic examination [6]. The size and shape ofhaustoria isolated from stem rust infections of wheat were similar to those reported here for leaf rust infections. The rigidity of the haustorial walls, the retention of haustorial necks and the presence of neckbands were noted [6, 16]. Electron microscopy of the outer surface of haustoria from Uromyces viciae-fabae showed that it was covered by a fibrillar layer representing the extrahaustorial matrix [6], as suspected in some of the preparations from Puccinia recondita. REFERENCES 1. B u s h n e l l WR. 1972. Physiology of fungal haustoria. Annual Review of Phytopathology 10 : 151-176. 2. C h a r d J M , Gay JL. 1984. Characterisation of the parasitic interface between Erisyphe pisi and Pisum sativum using fluorescent probes. Physiological Plant Pathology 25: 259-276. 3. C h o n g J, H a r d e r DE, R o h r i n g e r R. 1985. Cytochemical studies on Puccinia graminis f. sp. tritici in a compatible wheat host. I. Walls of intercellular hyphal cells and haustorium mother cells. Canadian Journal of Botany 63: 1713-1724. 4. C h o n g J, H a r d e r DE, R o h r i n g e r R. 1986. Cytochemical studies on Puccinia graminis f. sp. tritici in a compatible wheat host. II. Haustorium mother cell walls at the host cell penetration site, haustorial walls, and the extrahaustorial matrix. Canadian Journal of Botany 64: 278-287. 5. Gil F, Gay JL. 1977. Ultrastructural and physiological properties of the host interfacial components of haustoria of Erysiphe pisi in vivo and in vitro. Physiological Plant Pathology 10: 1-12. 6. H a h n M, M e n g d e n K. 1992. Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities. Protoplasma 170:95-113. 7. H a r d e r DE. 1989. Rust fungal haustoria--past, present and future. Canadian Journal of Plant Pathology 11: 91-99. 8. H a r d e r DE, C h o n g J. 1984. Structure and physiology ofhaustoria. In: Bushnell WR, Roelfs AP, eds. The Cereal Rusts Vol 1. Orlando: Academic Press, 431-476. 9. Harder DE, Chong J, Rohringer R, Mendgen K, Schneider A, Welter K, Knauf G. 1989. Ultrastructure and cytochemistry of extramural substances associated with intercellular hyphae of several rust fungi. Canadian Journal of Botany 76: 2043-2051. 10. H e a t h MC. 1989. In vitro formation of haustoria of the cowpea rust fungus, Uromyces vignae, in the absence of a riving plant cell. I. Light microscopy. Physiological and Molecular Plant Pathology 35: 357-366. 11. H e a t h MC. 1990. In vitro formation of haustoria of the cowpea rust fungus, Uromyces vignae, in the absence of a living plant cell. II. Electron microscopy. Canadian Journal of Botany 68: 278-287. 12. H u a ' a n Y, S i v a s i t h a m p a r a m K, O ' B r i e n PA. 1991. An improved technique for fluorescence staining of fungal nuclei and septa. Australasian Plant Pathology 20 : 119-121. 13. K a n g Z, R o h r i n g e r R, C h o n g J, H a b e r S. 1991. Microwave fixation of rust-infected wheat leaves. Preservation of fine structure and detection of cell surface antigens, [ectin- and sugar-binding sites. Protoplasma 162: 27-37. 14. Littlefield L J, H e a t h MC. 1979. Ultrastructure of Rust Fungi. New York: Academic Press. 15. P r u s k y D, D i n o o r A, J a c o b y B. 1980. The sequence of death of haustoria and host cells during the hypersensitive reaction of oat to crown rust. Physiological Plant Pathology 17: 33-40. 16. T i b u r z y R, M a r t i n s EMF, R e i s e n e r HJ. 1992. Isolation of haustoria of Puccinia graminis f. sp. tritici from wheat leaves. Experimental ~Iyeology 16: 324-328.
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PItvsiological and Molecular Plant Pathology 11993/ 42, 337 343
Isolation of haustoria from wheat leaves infected by the leaf rust fungus L. C. CANTRILL and B.J. DEVERALL D@aHment of Cr@ Sciem:e.~, t 'niversity of Sydney, . V.S. [ I ". 2006, Australia .lccepted Jar publication .l larch 19931
A proeedure was developed to isolate haustoria from wheat leaves infected by Puccinia recondita. Homogenization of heavily inR'cted leaves in an isolation medium ,.,.'as followed bv a period of sedimentation. 'fhe resulting suspension,,,,'assubjected to mild centrifugation and the pellet was separated further by centrifugation on a density gradient. One laver ',','asselected and washed before examination by optical microscopy. Two types of fungal structure were distinguishcd by their shape, size and cytoplasmicactivity and with a nuclear stain and Icctin probcs. One type was identified as hyphae and the other as haustoria. The rcasons for identification arc discussed in relation to literature on isolated haustorial complexes fi'om powdery mildew fungi and on clcctron microscopic examination of sections of rust haustoria in wheat leaves.
INTRODUCTION Haustoria are the only structures of rust and powdery mildew fungi that come into direct contact with the host protoplasm [1] and for this reason they are likely sites for the initiation of mechanisms of resistance and specificity. Access to haustoria for physiological and molecular studies is needed. I n rust fungi, the haustoria are e m b e d d e d deeply in the host tissue and m u c h has been written c o n c e r n i n g the difficulties thus posed in working with them [7]. In vitro formation of haustoria [10, I1 ] a n d isolation and purification of haustoria or haustorial complexes from the leaf [5] offer approaches in yielding haustoria that are free of host tissue. T h e a p p r o a c h of isolating rust haustoria and hyphae from leaves has been used here along with methods for confirming the n a t u r e of the fungal parts so obtained. Detailed ultrastructural and cytochemical analyses of the hyphae and haustoria of the wheat stem rust fungus have been achieved using lectin stains applied to c o n v e n t i o n a l electron microscopy preparations [3, 4 ]. A model of the c o m p o n e n t layers in rust fungal walls has been derived from the use of these stains. C h o n g et al. [4] represented haustorial walls with two layers, both of which contained chitin. H y p h a l walls (containing four layers) and haustorial m o t h e r cell walls (containing up to six layers) appeared to possess no chitin in outer layers [3], but carried surface residues of an a-linked glucose or m a n n o s e which were not detected in haustorial walls. T h u s a possible cytochemical method for differentiating haustorial surfaces from hyphal surfaces was indicated. Recent i m p r o v e m e n t s to p r e p a r a t i o n techniques for electron microscopy have shown 0885-5765/93/050337 + 07 $08.00/0
~) 1993 Academic Press Limited
338
L.C. Cantrill and B. J. Deverall
the cytochemistry of rust fungal walls to be even more complex, involving previously undetected lectin binding sites [13] and variable lectin binding in the extramural environment [9]. While such improvements refine the knowledge of non-specific lipids, polysaccharides and glycoproteins in and around the walls, the results caution against reliance upon lectin staining for distinguishing haustorial and hyphal surfaces. MATERIALS AND METHODS
Infected plants Seeds of Triticum aestivum L. cv. Little Club (10 per 12"5-cm pot in 650 g of a peat/river sand mixture with complete fertilizer) were germinated at approximately 22 °C under 16-h photoperiods (250 p.Em -2 s-1 at soil level). At 8 days, primary leaves of 30 plants were inoculated with 100 mg of washed uredospores of Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. & Henn. (race 104-2,3,6,7) by applying a spore/talc mixture (6: 10) using a paint brush. Plants were incubated in a dark humid chamber for 18 h at 23 °C (overnight) and then were returned to the conditions of seed germination for 7-14 days.
Phase contrast microscopy Heavily infected primary leaves (1"5 g) were homogenized for l min in 10 ml of isolation medium (0-1 M sucrose/0"01 M phosphate buffer pH 6"7, adapted from Gil & Gay [5]) using a Sorvall Omnimix set at speed 6. The homogenate was fhered through muslin and allowed to stand for 30 min in order to remove spores and other large material. The filtrate was then decanted from the precipitate and saved. Homogenization of retained debris was repeated three times (speed set successively to 6, 7 and 8) with further filtering and collection of filtrate after each speed increment. Each sample of filtrate was centrifuged at 800 g for 10 min. The supernatant was discarded and the pellet resuspended in 2 ml isolation medium. Drops of the suspension were placed on slides and examined with phase contrast on a Leitz Dialux microscope. Fluorescence of nuclei The cellular contents of the fungal material in resuspended pellets were examined using bisbenzimide H33258 fluorochrome (Hoechst). The dye was prepared by adding 0"6 ml of a stock solution (20 mg of Hoechst 33258 in 25 ml of distilled water) to 50 ml of isolation medium (adapted from Hua'an [12]). Dye solution (1 ml) was added to 1 ml offungal suspension. Mounts of whole mycelia were also prepared for comparison with the homogenized material. Squares (5 x 5 mm) of infected wheat leaf were incubated for 3 h in a macerating solution (0"5 °/o pectinase, 1"0 °/o hemicellulase, 2"0 °/o cellulase in a 10% sorbitol solution) and teased apart on a slide in a drop of dye solution. Both preparations were examined under UV illumination using a Leitz Orthoplan microscope fitted with a 200 W high-pressure mercury lamp and Leitz type A filter blocks. Fluorescent lectins Suspensions of fungal material were prepared as for phase contract microscopy. Each 2 ml suspension was layered onto an 8 ml 10-20 % continuous Ficoll 400 (Pharmacia) gradient made up in isolation medium and centrifuged for 5 min at 100 g. The middle
Isolation of haustoria
339
greenish-white layer of each gradient was collected and washed by diluting with isolation medium and centrifuged at 800 g for 10 min. The supernatant was discarded and the washing step repeated on the pelleted material. The final pellets were resuspended in 1 ml of the appropriate agglutinin buffer and 1 ml of stock agglutinin was added. The surfaces of the extracted fungal material were examined with the aid of either concanavalin agglutinin (ConA) or wheat germ agglutinin (WGA), each labelled with ftuorescein isothiocyanate (FITC). FITC--ConA was dissolved in a solution of 0"01 M Tris-HC1, 0"15 M NaC1, 1 mM CaCI~, and 1 mM MnC12 at p H 7"2 to make a final concentration of 200 lag ml -x in the fungal suspension. F I T C - - W G A was dissolved in 0-1 M phosphate buffer p H 7"2 to make a final concentration of 100 lag m1-1 in the fungal suspension (adapted from Chard & Gay [2]). Drops of the stained fungal suspension were mounted on slides and fluorescence was observed under UV with Leitz type I and 12 filter blocks. RESULTS
Homogenization released a mixture of fragments into the isolation medium (Fig. 1). Partial purification of the crude extract on a Ficoll 400 gradient eliminated most of the contaminating chloroplasts and removed the majority ofuredospores and larger fungal material. Two classes of structure remained and these were identified as hyphal fragments and haustoria for the following reasons. Hyphal fragments (Figs 1 and 4) were 4"5-5 lam wide and irregular in shape along
F[o. 1. Crude homogenatetreated with FITC-WGA ( 1O0 lag ml-x) showingbrightly fluorescent haustoria (H), chloroplasts (cl) and a large fragment of hypha (h) with septum (s).
340
L.C. Cantrill and B. J. Deverall
their axes, being periodically septate and branched. The ends of the fragments were roughly sheared. No cytoplasmic particles were observed under phase contrast and no Hoechst fluorochrome staining bodies were seen under UV illumination. The walls of hyphal fragments fluoresced lightly under UV illumination after staining with F I T C - C o n A or F I T C - W G A . The other class of fragments, identified as haustoria, were elongate (20-45 gm long, 5-10 gm wide) with rounded ends (Fig. 2). The outer walls were entire and there were
FIG. 2. Isolated haustorium under phase contrast showing neck-like projection (n) and cytoplasmic particles (cp). no septae or branches. Each haustorium bore a single rigid neck-like projection. The majority ofhaustoria viewed with phase contrast had transparent walls through which rapidly streaming cytoplasmic particles (0"5 gm diameter) were observed (Fig. 2). In some haustoria the path of the particles was obscured by an amorphous material attached to the walls. Streaming rates were reduced or stopped during further processing on Ficoll gradients and washing by centrifugation. However, unpurified haustoria continued cytoplasmic streaming for several hours after isolation. After staining with the Hoechst reagent the haustoria were seen to contain single 3 gm diameter spheres that fluoresced bright blue under UV illumination [Fig. 3(b)]. Walls ofhaustoria also fluoresced strongly in areas, usually at one end, when treated with the labelled lectins (Figs 1, 4). The rigid neck-like projection (Figs 2, 3, 4) was located at one end of the haustorium, usually the end that fluoresced least brightly with lectin stains. The projections often fluoresced with either of the two lectin probes (Fig. 4). Under phase contrast (Fig. 2) projections were revealed to be composed of two parallel walls (up to 3-5 gm long) which were continuous with the walls of the main haustorial body. The projections were 1 gm wide where they joined the body, narrowing halfway to 0"5 gm. Hand sections of infected leaves contained intercellular haustoria of the same shape and size as those released by homogenization. Similar haustoria were observed
Isolation of haustoria
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341
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10urn FzG. 3. (a) Bright field view ofhaustorium showing neck-like projection (n), as well as cytoplasmic particles and a large central vacuole (v). (b) Same haustorium treated with Hoechst 33258 fluorochrome and illuminated with UV showing faintly fluorescent walls and brightly fluorescent nucleus (n).
Flo. 4. Haustorium (H) and hyphal fragment (h) treated with FITC-ConA (200 lag ml-t). The neck-like projection (n) is highly visible due to staining with the lectin probe.
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342
L.C. Cantrill and B. J. Deverall
attached to mycelia released by enzymic digestion of pieces of infected leaves. The nuclei in the haustoria were similar in size and location to the spheres that fluoresced with Hoechst reagent in haustoria in homogenized material. DISCUSSION
Haustoria separated from their host by homogenization were identical in shape to haustoria observed in hand sections and to those attached to whole mycelia released by enzymic digestion of host tissue. The dimensions of the host-free haustoria and of their necks also compared favourably with those shown in sections prepared for electron microscopy [8, 14, 15]. Consistency in size, shape, staining patterns with labelled lectins and possession of neck-like projections on the majority of the isolated haustoria indicated that large numbers of uniform rust haustoria could be separated from wheat leaves by homogenization. The usefulness of these separated haustoria in further research is yet to be determined. Observations of streaming particles within the haustorial cellular space for several hours after isolation indicated that haustoria could be separated with cytoplasmic contents intact. The technique o f H u a ' a n et al. [12] revealed that the cytoplasm of hostfree haustoria contained fluorescent blue nuclei under UV and that these were identical to nuclei observed in hyphae and haustoria of whole mycelia. In contrast, streaming cytoplasm and stainable nuclei in hyphae were lost, probably after breakage or exposure of the protoplast to the isolation medium through wall gaps up to 5 lain wide. Earlier leakage of cytoplasm from haustoria may have been slowed by the narrowness of the space within the neck (0"5 lam) and the length of this narrow channel (3 lam). Gil & Gay [5] explained cytoplasmic retention in powdery mildew haustoria as a consequence of an amorphous plug in the septal pore between the neck and haustorial body and attributed durability to the presence of an extrahaustorial membrane. While rust haustoria do not possess a septum, blockage of the neck space by debris may occur during homogenization. At the same time it is unclear whether an extrahaustorial membrane is present or not. Amorphous material observed on the surface of some haustoria may have been a remnant of the extrahaustorial matrix. The lectin-binding properties of the host-free haustoria produced some conflicting results. From the model of stem rust haustoria described by Chong et al. [3, 4], it might be predicted that only WGA binding sites would be detected if the extrahaustorial membrane and matrix were totally absent. Alternatively, ConA binding sites might indicate the presence of these features. Under the conditions used here, both lectins were observed to bind to the haustoria in a similar pattern. Hyphal fragments also varied in the intensity of fluorescence. This may reflect differences in wall chemistry between leaf and stem rust fungi or differences between fresh and fixed material and the relative preservation and availability of active binding sites. Doubts have also been expressed concerning the reliability of F I T C - W G A and its specificity for chitin [10]. Changes in wall chemistry over time have also been noted, with a loss of ConA binding in hyphae found in sporogenous zones [9] and a changing affinity for ConA and WGA in haustoria as they mature [4]. The variability in lectin binding may have been a consequence of the advanced infections used here and the resultant mix of ages and developmental stages released by homogenization.
Isolation of haustoria
343
After this paper was submitted, the isolation of haustoria of the stem rust fungus of wheat (Puccinia graminis f. sp. tritici) was reported [6, 16] and also of haustoria of three other rust fungi, Puccinia sorghi and two species of Uromyces [6]. The methods involved homogenization of heavily-infected leaves as here, but they were followed by centrifugation on sucrose density gradients [16] or affinity chromatography of ConAbound extracts [6]. Viability of haustoria was recorded to be high according to exclusion of methylene blue stain [16] and to electron microscopic examination [6]. The size and shape ofhaustoria isolated from stem rust infections of wheat were similar to those reported here for leaf rust infections. The rigidity of the haustorial walls, the retention of haustorial necks and the presence of neckbands were noted [6, 16]. Electron microscopy of the outer surface of haustoria from Uromyces viciae-fabae showed that it was covered by a fibrillar layer representing the extrahaustorial matrix [6], as suspected in some of the preparations from Puccinia recondita. REFERENCES 1. B u s h n e l l WR. 1972. Physiology of fungal haustoria. Annual Review of Phytopathology 10 : 151-176. 2. C h a r d J M , Gay JL. 1984. Characterisation of the parasitic interface between Erisyphe pisi and Pisum sativum using fluorescent probes. Physiological Plant Pathology 25: 259-276. 3. C h o n g J, H a r d e r DE, R o h r i n g e r R. 1985. Cytochemical studies on Puccinia graminis f. sp. tritici in a compatible wheat host. I. Walls of intercellular hyphal cells and haustorium mother cells. Canadian Journal of Botany 63: 1713-1724. 4. C h o n g J, H a r d e r DE, R o h r i n g e r R. 1986. Cytochemical studies on Puccinia graminis f. sp. tritici in a compatible wheat host. II. Haustorium mother cell walls at the host cell penetration site, haustorial walls, and the extrahaustorial matrix. Canadian Journal of Botany 64: 278-287. 5. Gil F, Gay JL. 1977. Ultrastructural and physiological properties of the host interfacial components of haustoria of Erysiphe pisi in vivo and in vitro. Physiological Plant Pathology 10: 1-12. 6. H a h n M, M e n g d e n K. 1992. Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities. Protoplasma 170:95-113. 7. H a r d e r DE. 1989. Rust fungal haustoria--past, present and future. Canadian Journal of Plant Pathology 11: 91-99. 8. H a r d e r DE, C h o n g J. 1984. Structure and physiology ofhaustoria. In: Bushnell WR, Roelfs AP, eds. The Cereal Rusts Vol 1. Orlando: Academic Press, 431-476. 9. Harder DE, Chong J, Rohringer R, Mendgen K, Schneider A, Welter K, Knauf G. 1989. Ultrastructure and cytochemistry of extramural substances associated with intercellular hyphae of several rust fungi. Canadian Journal of Botany 76: 2043-2051. 10. H e a t h MC. 1989. In vitro formation of haustoria of the cowpea rust fungus, Uromyces vignae, in the absence of a riving plant cell. I. Light microscopy. Physiological and Molecular Plant Pathology 35: 357-366. 11. H e a t h MC. 1990. In vitro formation of haustoria of the cowpea rust fungus, Uromyces vignae, in the absence of a living plant cell. II. Electron microscopy. Canadian Journal of Botany 68: 278-287. 12. H u a ' a n Y, S i v a s i t h a m p a r a m K, O ' B r i e n PA. 1991. An improved technique for fluorescence staining of fungal nuclei and septa. Australasian Plant Pathology 20 : 119-121. 13. K a n g Z, R o h r i n g e r R, C h o n g J, H a b e r S. 1991. Microwave fixation of rust-infected wheat leaves. Preservation of fine structure and detection of cell surface antigens, [ectin- and sugar-binding sites. Protoplasma 162: 27-37. 14. Littlefield L J, H e a t h MC. 1979. Ultrastructure of Rust Fungi. New York: Academic Press. 15. P r u s k y D, D i n o o r A, J a c o b y B. 1980. The sequence of death of haustoria and host cells during the hypersensitive reaction of oat to crown rust. Physiological Plant Pathology 17: 33-40. 16. T i b u r z y R, M a r t i n s EMF, R e i s e n e r HJ. 1992. Isolation of haustoria of Puccinia graminis f. sp. tritici from wheat leaves. Experimental ~Iyeology 16: 324-328.
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